Neal, 1984.pdf

EXPERIMENTALPARASITOLOGY 57, 269-273(1984)

Leishmania major: Culture Media, Mouse Strains, and Promastigote Virulence and Infectivity

R. A. NEAL Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel Street,

London WCIE 7HT, England, U.K.

(Accepted for publication 25 January 1984)

NEAL, R. A. 1984. Leishmania major: Culture media, mouse strains, and promastigote virulence and infectivity. Experimental Parasitology 57, 269-273. Promastigotes of Leish- mania major were isolated from an infected mouse in two media, blood agar and Schneider’s medium + 30% fetal calf serum, and maintained continuously for over 1 year. Infectivity studies in two strains of mice, outbred CD1 strain and inbred BALB/c strain, showed that promastigotes grown in Schneider’s medium maintained infectivity to BALB/c mice throughout the period of cultivation. Infectivity to CD1 strain mice was progressively lost. Promastigotes grown in blood agar medium, however, lost infectivity to both strains of mice at a faster rate than promastigotes grown in Schneider’s medium.

INDEX DESCRIPTORS: Leishmaniu major; Protozoa, parasitic; Promastigote; Culture in vitro; Mice, CDl, BALB/c; Virulence; Infectivity.

INTRODUCTION

Previous studies on the chemotherapy of cutaneous leishmaniasis using the promastigotes of Leishmania major showed that, although the promastigotes lost infectivity with continuous cultivation, the organisms remained infective to mice up to the 10th week of cultivation (Neal 1964).

With the recent development of new media for the growth of Leishmania species based on insect cell tissue culture (Childs et al. 1978; Hendricks et al. 1978), it was decided to determine the duration of infectivity in the new media. Once established, growth of L. major was equally good in Schneiders’ medium or blood agar. Thus, infectivity comparisons were made over a period of more than a year using the original strain of mice used in previous studies and the susceptible inbred mouse strain BALB/c (Howard et al. 1980).

MATERIALS ANDMETHODS

The strain previously known as Leishmoniu tropica major has been maintained continuously since 1959, and is now known as Leishmanin major (Liverpool

reference number, LV39). Its history is given in Neal (1964).

Schneider’s medium was prepared commercially (GIBCO) and 30% heat-inactivated fetal calf serum (Wellcome Diagnostics) was added before use. Five milliliters of the complete medium was pipetted into 25-cm2 plastic tissue culture flasks (Coming).

The blood agar medium was prepared by adding 10% v/v defibrinated fresh rabbits blood to molten bac- teriological nutrient agar. The agar was cooled to about 45 C before the addition of blood. About 5 ml of the mixture was transferred to 5-0~ medical flat screw-capped bottles, and solidified on the flat side of the bottle. The blood agar medium was completed by the addition of 5 ml of glucose saline. Blood agar medium was used within 2 weeks of preparation. All media were stored at 4 C.

The L. major cultures originated from the nonul- cerated lesion, about 3 mm diameter, of a mouse inoculated 12 days previously with L. major promastigotes. Portions of the lesion containing amastigotes were inoculated into blood agar medium and Schnei- der’s medium + 30% fetal calf serum. The liquid phase of these media contained about 10,000 I.U. or pg of benzylpenicillin (as sodium salt) and strepto- mycin (as sulphate salt), respectively. Cultures were incubated at 26 C. After the primary cultures, no further antibacterial compounds were added during the period of cultivation. At no time were the cultures contaminated with fungi or bacteria. The cultures were maintained by subcultivation at the end of the loga-

269 0014-4894184 $3.00 Copyright 8 1984 by Academic Press, Inc. All rights of reproduction in any fom, reserved.

270 R. A. NEAL

rithmic growth phase, every 3 to 4 days about lo6 promastigotes being transferred to fresh medium. By the end of the series of experiments, the cultures had reached subculture nos. 111 (blood agar) and 110 (Schneider’s).

Male mice, weighing about 20 g, were obtained from commercial breeders; outbred strain CD1 (Charles Rivers) and inbred strain BALB/c (Bantin & Kingman). After inoculation, the mice were divided into groups of 10, and fed with commercial mouse food and given water ad libirum. The animal room was not air conditioned but was situated on the cooler north side of the building and heated to maintain a room temperature of about 23 C.

The inoculum was prepared from 72-hr cultures in either blood agar or Schneider’s medium. The promastigotes were pooled and counted with a haemo- cytometer, and the density was adjusted to give 10s promastigotes/ml. Rosettes of flagellates were dis- persed by agitating on a laboratory shaker. One-tenth milliliter of promastigote suspension was inoculated subcutaneously on the dorsal body surface, about 10 mm. anterior to the root of the tail. This area was closely clipped to remove hair before inoculation, but was not clipped again during the experiment.

The inoculated mice were inspected for lesion development every 7 days, starting 15 to 18 days after inoculation. When lesions were observed, each lesion was measured in two directions at right angles to each other. The mean of the two measurements was re- corded. Observations were continued for about 3 months in each experiment. In one experiment, impression smears were prepared from the lesion or from the skin at the site of inoculation. The impression smears were fixed with methanol and stained with Giemsa stain, Smears were examined microscopically for the presence of amastigotes.

RESULTS

In the primary Leishmania major cultures, heavy promastigote growth occurred within 3 days in the blood agar medium, whereas, in Schneider’s medium, initial promastigote growth occurred more slowly, requiring 8 to 11 days to reach maximum growth. After this initial period, the promastigote growth was similar in both media, reaching a maximum concentration of between lo7 and lo8 flagellates/ml within 4 days.

The two strains of mice were inoculated with promastigotes from the two media after 3, 11, 23, and 57 weeks of continuous in vitro cultivation. The graphs of lesion de-

velopment are shown in Figs. 1, 2, 3, and 4. The lesions in BALB/c mice developed quicker and to a larger size than those in CD1 mice. With continued cultivation in vitro, lesion development was progressively slower with promastigotes grown in both media. After 3 weeks cultivation (Fig. l.), lesion development was that expected for the two strains of mice, and no difference was seen between the two media. After 11 weeks cultivation (Fig. 2.), lesion development from promastigotes inoculated into BALB/c mice were identical, but lesions in CD1 mice were not progressive. Again no difference was seen between the two media. However, after 23 weeks in continuous cultivation, progressive lesions were observed with BALB/c mice inoculated with promastigotes grown in Schnei- der’s medium, while there was no signifi-

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CD1 mica, 8chndden medium 9alblc mica, blood a9m mdium Bdbh mice, Sdmeiden mdium

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DAY AFTER INOCULATION

FIG. 1. Development of mouse lesions due to inoculation of Leishmania major promastigotes grown in two culture media for 3 weeks.

,?hhmaniu major: VARIATIONS IN VIRULENCE 271

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DAV AFTER INOCULATION

FIG. 2. As Fig. 1 but Leishmania major grown in vitro for 11 weeks.

cant lesion growth in CD1 mice inoculated with blood agar-grown promastigotes. CD1 mice inoculated with Schneider’+grown promastigotes, and BALB/c mice inocu-

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lated with blood agar promastigotes, were intermediate in the rate of lesion development. The last observations were made after 57 weeks of continuous cultivation (Fig. 4). At this time, only promastigotes grown in Schneider’s medium and inoculated into BALB/c mice showed lesion development, though it was slower than in previous experiments.

Two groups of 10 mice, of BALB/c and CD1 strains, were separated from the batch of mice used in the final experiment. These mice were inoculated with freshly isolated promastigotes grown for 3 weeks in blood agar medium. The cutaneous lesion developed at a similar rate to those shown in Fig. 1, thus demonstrating that the failure to in- fect by long-term growth in vitro (see Fig. 4) was due to the parasites and not the host.

The differences between the three factors, mouse strain, culture medium, and length of in vitro cultivation, is also shown by the difference in infectivity (Fig. 5). This shows that there was no loss of infectivity by promastigotes maintained in Schneider’s medium and inoculated into BALB/c mice. While the most rapid loss of infectivity was with promastigotes grown in blood agar and inoculated into CD1 mice, other combina- tions were intermediate in their infectivity.

212 It. A. NEAL

10 20 30 a 50 60 WEEKS IN CONTlN”O”S C”LTIY*TION

FIG. 5. Proportion of mice infected by inoculation of Leishmania major promastigotes grown in vitro in two culture media.

Metastatic spread of the parasites was not observed during the period of observation.

DISCUSSION

One explanation of the loss of Leish- mania major infectivity could be a change in growth rate in vitro, assuming that this parallels the intracellular growth rate of amastigotes. Although no direct growth- curve observations were made, the yield of promastigotes for inoculation was not no- ticeably different at any time. Therefore, if it did vary, it is likely that any difference was small.

Loss ,of virulence by in vitro growth of microorganisms is a well-known phenom- enon. Amongst ‘the protozoa, it has been well described for Entamoeba histolytica (Vincent and Neal 1960; Martinez-Palomo 1982). Previous work showed that L. major promastigotes had lost infectivity to mice but were still infective to man (Adler 1961). The earlier work, however, employed outbred strains of mice which may have been more resistant and less uniform in their response to inoculation with promastigotes. It may be argued that, the susceptibility to man may be more similar to infectivity of BALB/c mice although, in these mice, the lesions are not self-limiting. More

recent work on infectivity of L. donovani to hamsters has indicated a loss of infectivity by continuous cultivation of promastigotes in vitro (Semprevivo et al. 1981).

It is possible that the two media selected different subpopulations, but this requires further studies with cloned material. Handman et al. (1983) showed the presence of noninfective clones in their strain of L. major. The present studies show that there are at least two factors contributing to the expression of virulence, the isolation and culture in particular media and the susceptibility of the mouse.

The biochemical studies on the variation of virulence has given conflicting results. Using cloned material, Handman et al. (1983) were unable to correlate the presence or absence of infectivity of L. major with radiolabelled cytoplasmic or membrane proteins, lectin binding, binding to monoclonal antibodies, or analysis of isolated kinetoplast DNA. There was evidence suggesting a correlation with infectivity in vitro to BALB/c macarophages. However, Giannini et al. (1981) were unable to show a difference in attachment of promastigotes to macrophages between infective and noninfective promastigotes of L. donovani, and postulated a difference in the post attachment stages. The situation on correlation of infectivity with lectin binding to promastigotes may be different with L. bruziliensis, since Dawidowicz et al. (1975) observed a difference with binding of Ricinus com- murk agglutinin (RCA). The degree of RCA binding to L. donovani promastigotes was shown by Doran and Herman (1981) to be less with organisms cultured for a longer period. It would be valuable to examine the present L. major cultures for macrophage infectivity and lectin binding.

The original purpose of these experiments was to check the length of time that infectivity and virulence was maintained during continuous cultivation in the new culture medium. From the results, it is clear that Schneider’s medium can be substituted

Leishmania major: VARIATIONS IN VIRULENCE 273

for blood agar and the Leishmania major promastigotes remain infective to BALBlc mice for prolonged periods.

REFERENCES ADLER, S. 1961. Infectivity of a strain of Leishmaniu

donovani after prolonged culture. Bulletin ofthe Re- search Council of Israel, Section B, 9E, 166.

CHILDS, G. E., FOSTER, K. A., AND MCROBERTS, M. J. 1978. Insect cell culture media for cultivation of New World Leishmania. International Journal

for Parasitology 8, 255-258. DAWIDOWICZ, K., HERNANDEZ, A. G., AND INFANTE,

R. B. 1975. The surface membrane of Leishmaniu. I. The effects of lectins on different stages of Leish- mania braziliensis. Journal of Parasitology 61,950- 953.

DORAN, T. I., AND HERMAN, R. 1981. Characterization of populations of promastigotes of Leishmaniu donovani. Journal of Protozoology 28, 345-350.

GIANNINI, M. S. H., D’ALESANDRO, P. A., GARCIA, C. R., AND SARAIVA, E. M. B. 1981. Failure of ham- ster macrophages to discriminate between infective and noninfective promastigotes of Leishmnnia donovani during attachment in vitro. Infection and Zm- munity 34, 629-632.

HANDMAN, E., HOCKING, R. E., MITCHELL, G. F., AND SPITHILL, T. W. 1983. Isolation and character-

ization of infective and non-infective clones of Leishmania tropica. Molecular and Biochemical Parasitology 7, 1 1 1 - 126.

HENDRICKS, L. D., WOOD, D. E., AND HAJDUK, M. E. 1978. Haemoflagellates: commercially available liquid media for rapid cultivation. Parasitology 76, 309-316.

HOWARD, J. G., HALE, C., AND CHAN-LIEW, W. L. 1980. Immunological regulation of experimental cutaneous leishmaniasis. 1. Immunogenetic aspects of susceptibility to Leishmania tropica in mice. Para- site Zmmunology 2, 303-314.

MARTINEZ-PALOMO, A. 1982. The biology of Enta- moeba histolytica. In “Tropical Medicine Research Studies Series” (K. N. Brown, ed.), Vol. 2, Wiley/ Research Studies Press, Chichester, U.K.

NEAL, R. A. 1964. Chemotherapy of cutaneous leishmaniasis: Leishmania tropicn infections in mice. Annals of Tropical Medicine and Parasitology 58, 420-430.

SEMPREVIVO, L. H., YUSUF, J. N., AND HONIGBERG, B. M. 1981. Changes in the growth rate and infectivity df Leishmania donovani subjected to various laboratory procedures. Zeitschrift fiir Parasiten- kunde 65, 43-51.

VINCENT, P., AND NEAL, R. A. 1960. Duration of in- vasiveness of Entamoeba histolytica maintained in vitro. Parasitology 50, 449-452.

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Neal, 1984.pdf